The cationic biopolymer ε-polylysine (ε-PL) is a potent food-grade antimicrobial that is highly effective against a range of food pathogens and spoilage organisms. In compositionally complex systems like foods and beverages, cationic ε-PL molecules may associate with anionic substances, leading to increased turbidity, sediment formation, and reduced antimicrobial activity. In this study, we therefore characterized the interactions between cationic ε-PL and anionic pectins with different degrees of esterification (DE), and then investigated the influence of these interactions on the antimicrobial efficacy of ε-PL. The nature of the interactions was characterized using isothermal titration calorimetry (ITC), micro-electrophoresis (ME), and turbidity measurements. High (DE 61%), medium (DE 51%) and low (DE 42%) methoxyl pectins interacted with ε-PL molecules through electrostatic forces, forming either soluble or insoluble complexes with varying electrical charges, depending on the relative mass ratio of pectin and ε-PL. The interaction of pectin with ε-PL increased as the negative charge density on the pectin molecules increased, i.e., with decreasing DE. The antimicrobial efficacy of ε-PL against two acid resistant spoilage yeasts (Zygosaccharomyces bailii and Saccharomyces cerevisiae) decreased progressively in the presence of increasing levels of all three pectins. The lowest DE pectin reduced the antimicrobial efficacy of ε-PL at lower concentrations (pectin-to-ε-PL  4) than the highest DE pectin (pectin-to-ε-PL  20), which is likely due to strong electrostatic binding of ε-PL with low DE pectin reducing its interaction with anionic microbe surfaces. Our study provides knowledge that will facilitate the rational application of ε-PL as an antimicrobial in complex food systems.